Received May 17, 2012; Revision received May 25, 2012
The modern concepts of programmed cell death (PCD) in plants are
reviewed as compared to PCD (apoptosis) in animals. Special attention
is focused on considering the potential mechanisms of implementation of
this fundamental biological process and its participants. In
particular, the proteolytic enzymes involved in PCD in animals
(caspases) and plants (phytaspases) are compared. Emphasis is put on
elucidation of both common features and substantial differences of PCD
implementation in plants and animals.
KEY WORDS: apoptosis, caspase, programmed cell death, phytaspase

Multicellular organisms require the ability to eliminate excessive or
damaged cells that are formed both during normal development and in the
interaction of the organism with the environment, e.g. under stress
conditions leading to irreversible cell damage, as well as in infection
with pathogens. The cellular process directed to successive
extermination (suicide) of undesirable cells is known as programmed
cell death (PCD). The most studied (although not the only) form of PCD
in animals is apoptosis, which is characterized by a distinct set of
morphological and biochemical features [1]. A
crucial role in programmed suicide of animal cells belongs to caspases,
a family of highly specific cysteine proteinases that are activated in
apoptosis, introducing single breaks in molecules of a restricted set
of cellular proteins [2]. Caspases have exclusive
specificity of hydrolysis: they introduce break after an aspartic acid
residue (D) localized within a certain amino acid context. Directed
fragmentation of target proteins by caspases eventually leads to the
ordered death of the cell. And, contrariwise, inhibition of caspases
counteracts apoptosis.

In correspondence with the name “apoptosis” (in
Greek – the fall of the leaf), PCD also occurs in plants,
playing the same role as in animals. Plants use PCD both in the course
of development (for instance, during xylem formation, seed germination,
prevention of self-pollination, and senescence) and in response to
osmotic, thermal, and oxidative stresses and in defense from pathogens.
Like in animals, PCD in plants takes various forms [3, 4], but a series of common PCD
features can be traced in both kingdoms. These features include DNA
fragmentation, cytochrome c release from mitochondria, cell
shrinkage, generation of reactive oxygen species, exposure of
phosphatidylserine, etc. [5].

It is worth noting that molecular mechanisms of plant PCD are much less
studied than those of animal cell apoptosis. However, similar
morphological features of animal and plant PCD imply the existence of
similar fundamentals in the organisms of these two kingdoms used for
PCD. In this regard, it is intriguing and significant that caspases,
which generally fulfill PCD in animals, are absent in plants, as
evident from sequencing of plant genomes. At the same time, much data
suggests that inhibitors of animal caspases can suppress PCD
development in plants. In connection with this, in plant PCD activation
of unidentified caspase-like proteases is also observed, and these can
hydrolyze various peptide substrates of caspases [6]. These data suggest that PCD in plants involves
proteases that are functional analogs of animal caspases, but which are
structurally different from caspases.

In this review, we focus on similarity and difference in PCD in plants
and animals and give a modern view on plant proteases that might
fulfill the role of caspases in PCD in plants.

CASPASES – APOPTOTIC PROTEASES OF ANIMALS

Structure and properties of caspases. Animal caspases differ from
other proteases in a number of features. “Caspase” is an
acronym from cysteine-dependent aspartate-specific proteases.
Approximately 10 caspases having strict aspartate specificity of
hydrolysis and differing in the preferred recognition site motif have
been identified in mammals [7]. To avoid untimely
triggering of the cell death mechanism, caspases are synthesized and
stored in the cytoplasm as inactive precursors –
procaspases. Procaspases are activated through their processing when
the pro-domain is removed and the major part of the protein is cleaved
into two subunits: p20 and p10. The active enzyme is a homodimer, where
each monomer consists of one p20 chain and one p10 chain. The complex
forms two symmetric active sites [8]. The catalytic
dyad includes amino acid residues of the p20 chain and consists of an
active-site cysteine residue that is a part of the conservative
sequence QACXG and a histidine residue.

The absolute specificity of animal caspases to peptide bond hydrolysis
after the D residue has already become “the talk of the
town”. Caspases are very selective and usually make one, or
rarely two, breaks per protein, i.e. are not degrading but processing
proteases. This is due to the fact that caspases usually recognize in
the substrate a tetrapeptide sequence with an aspartic acid residue at
its C-terminus, after which the peptide bond is hydrolyzed (P1
substrate position). The preferred hydrolysis sequences for different
caspases are determined by the amino acid motif of the recognition site
[9, 10].

To date, about 400 caspase-cleaved substrates have been described (these
target proteins are presented in the CASBAH database, http://www.casbah.ie).
They include structural proteins, transcriptional and translational
regulator proteins, kinases, signaling pathway components, pathogen
proteins, etc. The limited proteolysis of cell proteins by caspases is
aimed at successive switching of cellular pathways from life to
programmed death. The notion that fragmentation of target proteins by
caspases leads to inactivation of proteins vitally important to the
cell and therefore results in its death is only partly true. Another
objective of caspase hydrolysis is activation of proapoptotic
mechanisms in a dying cell. So, fragmentation of the Bid protein from
the Bcl-2 family provides the formed Bid fragment (so-called tBid,
truncated Bid) the ability to be directed to mitochondria and to
promote cytochrome c release from the mitochondria, which leads
to a drastic increase in caspase activity in the cell (see below) [11, 12].

Another canonical example of activation through caspase hydrolysis is
CAD nuclease. In healthy cells, the activity of this nuclease is
suppressed due to its interaction with the inhibitor protein ICAD.
Caspase-3 activated during the induction of apoptosis introduces two
breaks in the ICAD molecule, which results in elimination of
inhibition, activation of the nuclease through dimerization, and,
finally, fragmentation of internucleosomes of cellular DNA with
formation of a DNA “ladder” so typical of apoptotic cells
[13-15].

However, it should be noted that the total number of caspase target
proteins is relatively low – about 2% of all proteins of
mammalian cells. Whether the hydrolysis of all targets by caspases is
necessary for apoptosis or some proteins are merely “innocent
bystanders” [16] is in most cases still an
open question needing further investigation.

Caspases are divided into two groups by their functions in the apoptotic
cascade. Initiator caspases (caspases-1, -2, -4, -5, -8, -9, -10, -11,
-12) are activated in response to proapoptotic or other stimuli and
participate in the processing (i.e. activation) of precursor proteins
of other caspases, thereby forming a cascade of proteolytic enzymes.
Effector, or executioner, caspases (-3, -6, -7) are activated by
upstream initiator caspases and hydrolyze various cell proteins (see
above), causing cell death [17, 18]. The structures of these enzymes are also
different in accordance with this division. Initiator caspases have an
extended pro-domain with one or two motifs responsible for the
interaction with adaptor molecules. These are so-called DED (death
effector domain) and CARD (caspase recruitment domain). Effector
caspases have a shorter pro-domain.

Caspases can be also divided into proapoptotic and proinflammatory.
Proapoptotic caspases (-2, -3, -6, -7, -8, -9, -10) are involved mainly
in implementation of PCD. Proinflammatory caspases (-1, -4, -5, -11,
-12) participate in the processing of cytokines during inflammation.
However, since the activation of proinflammatory caspases can provoke
apoptosis, this subdivision of caspases into groups is convenient but
conditional. At the same time, more and more data demonstrate that
caspases may be involved in different cellular processes unrelated to
apoptosis or inflammation. It has been shown that caspase-8
participates in proliferation of immune cells [19-22] and in cell
differentiation [23]. Caspase-3 is involved in
differentiation of the long-living cells of skeletal muscles,
osteoblasts, and neurons [24, 25]. A case in point is the involvement of
proinflammatory caspase-1 in the processing of precursors of
interleukins IL-1β and IL-18 [26, 27]. In addition, caspase-3 is able to process the
precursor protein of IL-16 [28].

Caspase activation mechanisms. Caspases are activated upon the
receipt of certain proapoptotic signals by the cell [29]. Two pathways of caspase activation during PCD
induction have been described. One is associated with a group of
transmembrane proteins, “death receptors”, which act as
surface sensors locating external ligands signaling about the need for
apoptosis. Among the best characterized “death receptors”
are the tumor necrosis factor receptor (TNFR1), as well as Fas, DR3,
TRAILR1 (TNF related apoptosis-inducing ligand receptor 1), TRAILR2,
etc. [30]. Upon binding of the respective ligands,
the death receptors multimerize with the formation of death-inducing
signaling complexes (DISC complexes). Adaptor proteins are recruited to
the DISC complex from the cytoplasmic side. For the Fas or TRAIL
receptors, for example, it is a Fas-associated DD (FADD) protein, which
is included in the complex via its C-terminal DD-domain, while its
N-terminal death effector domain (DED) interacts with the same domain
of caspase-8. Oligomerization of caspase-8 molecules in the DISC
complex is considered to trigger autocatalytic activation of the
caspase and, thereby, initiation of programmed cell death [17, 31]. Depending on the type
of cells, caspase-8 can activate executive caspases -3 and -7 by
cleaving the pro-domain, and this seems to be sufficient for apoptotic
cell death. In other cases, the signal received by caspase-8 may be
amplified via the mitochondrial apoptotic pathway [12].

The mitochondrial (“internal”) pathway is another pathway
leading to cell death. It is switched on in the case of internal cell
defects (DNA damage, various stresses, cytotoxic agents). Regulation of
this pathway involves a large group of proteins from the Bcl-2 family.
The latter includes both pro- and antiapoptotic proteins [32-39]. The perception of an
apoptosis-inducing signal activates the proapoptotic proteins of this
family, which form a Bak–Bax oligomeric complex in the outer
mitochondrial membrane. This results in formation of channels through
which cytochrome c is released from the mitochondria [40]. Cytochrome c, in turn, stimulates the
assembly of a complex named the “apoptosome” [41] and triggers a sequence of events leading to the
activation of procaspase-9. The apoptosome is a multi-protein complex
comprising the following proteins: Apaf-1, cytochrome c, and
dATP/ATP as a cofactor [42-44]. It serves as a “platform” for
procaspase-9 binding and dimerization, which, in turn, leads to the
autocatalytic processing of procaspase. This results in formation of
two subunits of caspase-9, p35 and p12, combined into active dimers [45]. After activation in the apoptosome, caspase-9
triggers the processing of caspases-2, -3, -6, -7, -8, and probably
caspase-1 [18, 46].

The two pathways of activation of apoptotic events are not independent.
The proapoptotic protein Bid is directly cleaved by caspase-8, and the
formed C-terminal fragment of this protein stimulates the release of
cytochrome c from mitochondria [12],
thereby increasing the apoptotic effect of the signal arriving through
external receptors.

Regulation of caspase activity. Since the decision whether
“to live or to die” is of vital importance for the cell and
for the organism as a whole, it would be strange if caspase activity
was not controlled in different ways. It is known that there is a
multistep system of caspase activity control [47].
The induction of apoptosis is accompanied by abrupt increase of
expression of the caspase genes [48-50]. The activity of caspases and, consequently, the
development of apoptotic events are also regulated by various kinase
signaling pathways. It has been shown, for example, that
phosphorylation of caspase-9 leads to inhibition of its activity [51, 52] and suppression of
apoptosis. In addition, animal cells contain endogenous inhibitor
proteins capable of regulating the activity of mature caspases in
vivo. The most significant of them are proteins of the IAP
(inhibitor of apoptosis) family [53, 54]. They can bind caspases and not only neutralize
the low level of caspase activity, but also create a barrier, above
which a drastic activation of the caspase cascade begins [55]. Proteins that derepress IAP-bound caspases have
also been characterized. Upon induction of PCD, these proteins (Smac,
HtrA2, and some other proteins) are released from the intermembrane
space of mitochondria with the aid of Bcl-2 proapoptotic proteins. Smac
is able to displace IAP from its complex with caspase, thereby
activating the apoptotic protease [56]. HtrA2
seems to act in an analogous manner. However, since HtrA2 is a protease
itself, it has another way of eliminating caspase inhibition, this time
irreversibly. HtrA2 can cleave most of the known IAPs, thereby
activating caspases [57, 58].
In addition, some viral proteins (baculovirus protein p35, cowpox virus
serpin CrmA) are able to inhibit caspases in the cells of the host
organism [59-61], which is
not very surprising because in many cases rapid cell death prevents
replication of the virus.

PROGRAMMED CELL DEATH OF PLANTS

Forms and manifestations of cell death. As mentioned above,
apoptosis in animals and PCD in plants have some similar morphological
features [3-5]. However, PCD
manifestations in plants have certain specificity. In some PCD models,
DNA fragmentation in plant cells is accompanied by formation of
extended DNA fragments but not an internucleosomal
“ladder”. A substantial difference between animals and
plants is also observed at the final stage of PCD development. In
animals, the dying cell forms apoptotic bodies that are instantaneously
phagocytized, thus allowing to avoid the lysis of dying cells and the
inflammatory response of the organism. In plants, phagocytosis of dying
cells is lacking not only due to the absence of professional
phagocytizing cells, but also due to the presence of rigid cellulose
walls separating the cells. The formation of apoptotic bodies has not
been observed in plants either. Therefore, apoptotic plant cells must
be eventually lysed and their contents are utilized.

The degree of cell wall degradation may vary depending on the type of
tissue formed. Deep degradation is observed during aerenchyma
formation, during leaf perforation formation, and when petals die off
[62-64]. As a result, an
empty space is left in the place of the dead cell. But every cloud has
a silver lining. The triggering of the PCD mechanism that leads to the
formation of aerenchyma (channels through which oxygen can be delivered
to the roots from the above-ground parts of a plant) allows some plants
(particularly rice) to survive on flooded soils. In other cases, the
cell wall remains intact as, e.g. in the cases of xylem formation,
rearrangement of leaf tissues, or fruit body formation [65, 66].

Recently an attempt has been made to classify plant cell death by a set
of typical morphological characters. According to this classification,
two main types of cell death are recognized: vacuolar cell death and
necrotic cell death [67]. Vacuolar cell death is
considered as a combination of autophagy performed by vacuoles and
accompanied by increase in their sizes, followed by the release of
hydrolases from the lytic vacuoles as a result of disruption of the
vacuolar membrane (tonoplast). At the same time, the morphology of cell
organelles and the integrity of cell plasma membrane are preserved till
the moment of tonoplast disruption. Such type of cell death takes days
and is typical of PCD occurring in the course of development of the
organism.

On the contrary, necrotic death is accompanied by rapid disruption of
the plasma membrane, shrinkage of the protoplast, disturbance of
mitochondrial function, accumulation of active oxygen forms, and
absence of characteristic features of vacuolar death. Necrotic death is
believed to occur under conditions of abiotic stresses.

However, there are many cases of plant PCD not falling within either of
the described categories. For example, hypersensitive response (HR) of
plant cells to infection by pathogens (see below) is a well-described
form of PCD but, at the same time, combines the signs of both vacuolar
and necrotic death. In addition, shrinkage of the protoplast may not
indicate disruption of the cell plasma membrane. For example, in one of
the described HR variants the cell plasma membrane remains intact in
spite of shrinkage of the protoplast [68], which
shows the absence of a direct relationship between these phenomena.

It is clear that the proposed classification based solely on
morphological features and assuming quite a number of non-classifiable
exclusions is tentative. Therefore, it would be advisable to have a
notion about the molecular mechanisms and basic components of the PCD
apparatus in plants for its improvement.

Some data of that kind have been obtained in the study of hypersensitive
response of plants. HR as a form of PCD is due to the fact that plants,
in contrast to animals, have no immune system that could neutralize
pathogens and infected cells. Therefore, plants use another strategy:
induction of suicide of infected and surrounding cells. This prevents
reproduction of the pathogen, on one hand, and creates a barrier of
dead cells separating the pathogen from healthy tissue, on the other
hand [69, 70]. The
morphological features of cell death during HR in many respects
coincide with those enlisted above, which are observed during PCD
induced by other stimuli. HR induction requires recognition by a
special protein of the plant (the R (resistance) gene product)
of the respective protein of the pathogen (the product of the so-called
avirulence (Avr) gene) [71, 72]. The R and Avr gene pairs can
encode various proteins, or these genes may be absent. In the case of
tobacco plants infected by the tobacco mosaic virus (TMV), the
resistance gene is the so-called N gene, which is present not in
all tobacco varieties. The product of this gene recognizes the viral
protein replicase (which in this case is the product of the avirulence
gene) and triggers HR. As a result, at the cost of death of a limited
number of cells, the plant prevents the development of viral infection.
Plants lacking the resistance gene do not respond to infection by
induced cell death (HR); as a result, the pathogen spreads over the
whole plant.

Vacuoles play a significant role in PCD that occurs not only during the
development of a plant, but also during HR induced by infection of
plants by viral, bacterial, or other pathogens. Two scenarios of PCD
development have been described. During viral infection, the tonoplast
is lysed with the release of the lytic enzymes of vacuoles into the
cytosol [73, 74]. This is of
biological significance because the overwhelming majority of plant
viruses reproduce just in the cytosol.

During bacterial or fungal infection, a pathogen is located outside the
plant cell, in the intercellular fluid (apoplast). Such pathogens
affect plant cells by means of so-called “effector”
proteins secreted by pathogens into plant cells. For controlling some
extracellular pathogens, the vacuolar membrane can be fused with the
plasma membrane, permitting the hydrolytic enzymes of the vacuole to be
released into the extracellular space [75].
Membrane fusion is induced by the interaction between the plant
R-gene product and the pathogen avirulence factor and ends not
only with neutralization of the pathogen, but also with induction of
PCD in the infected plant cells. The process of fusion of the vacuolar
and plasma membranes during PCD caused by certain pathogenic strains
was shown to require the functioning of plant cell proteasomes.
Inhibition of proteasome activity by peptide inhibitors suppresses
membrane fusion and the release of vacuolar proteins into the
intercellular space [75]. RNA silencing of any of
the genes encoding the subunits of Arabidopsis thaliana
proteasome also inhibits membrane fusion.

Proteasome activity is important not only for membrane fusion, but also
for the development of HR during bacterial infection. The authors [75] measured the percentage of dead cells (by their
ability to be stained with trypan blue and by the electrical
conductivity of tissues increasing during cell death) and thereby
showed that inhibition of the activity of any of the proteasome
subunits prevents HR development.

It is interesting that the peptide inhibitor of human caspase-3,
Ac-DEVD-FMK (see the next section for more detailed information about
the structure of peptide inhibitors of caspases), prevented HR in the
case of bacterial infection, which suggested to the authors [75] that membrane fusion requires an activity similar
to the activity of caspase-3, and this caspase-like activity might be
typical of the plant proteasome.

It should be noted that the fact that animal and yeast proteasomes
possess a caspase-like activity has been known for a long time [76]. Moreover, the animal proteasome inhibitor
Ac-APnLD-CHO (nL = norleucine) proved to be an inhibitor of the
plant proteasome subunit PBA1 as well, indicating the possible presence
of caspase-like activity in this subunit. The silencing of the
Arabidopsis PBA1 gene reduced the DEVDase activity found in
extracts, which did not contradict the above suggestion but, however,
was not strict evidence for it. The inhibition of other proteasome
subunits possessing trypsin- and chymotrypsin-like activities was
observed as well. Moreover, cell death was also suppressed by the
common proteasome inhibitor clasto-lactacystin β-lactone, as well
as by silencing of the genes of other proteasome subunits. It seems
that the determining factor in implementation of this type of PCD is
the activity of the proteasome as a whole but not the supposed
caspase-like activity of subunit PBA1. Hence, the application of
biotinylated inhibitor DEVD-FMK resulted in the inhibition of activity
of not only PBA1, but also, strange as it may seem, of other proteasome
subunits. Nevertheless, the PBA1 subunit bound the DEVD-FMK inhibitor.
However, the traditional proteasome inhibitor MG132 (LLL-CHO)
containing no D residue was also able to modify PBA1 [77]. It seems that the question whether any plant
proteasome subunit displays a specific caspase-like activity and
whether this activity is necessary for plant PCD implementation needs
further elucidation.

Approaches for detection of caspase-like plant proteases. In
spite of some similar features of PCD in animals and plants, the
question about the similarity of molecular mechanisms of PCD in the two
kingdoms is still open. For example, the absence of caspases (the key
apoptotic enzymes of animals) in plants is a striking example of
difference (at least technically) between animal and plant PCD.
Therefore, it is highly relevant to determine whether any plant
proteases perform the functions of caspases during PCD and, if so, what
these proteases are.

An argument in favor of the assumption that caspase-specific proteases
of plants exist and participate in PCD was the fact that the protein
inhibitors of animal caspases (baculovirus proteins p35 and Op-IAP),
which are produced in plants, were reported to prevent the development
of PCD. Transgenic tomato plants carrying the p35 gene proved to be
more resistant to toxin-induced PCD and towards infection with various
pathogenic fungi [78], while HR development in
transgenic (by the p35 gene) tobacco leaves caused by the
Pseudomonas syringae infection was partially suppressed [79]. In both cases transgenic plants, possessing the
gene of mutant protein p35, which is not an inhibitor of animal
caspases, had no antiapoptotic properties. The expression of the p35
gene in the embryonic callus of maize also suppressed PCD [80]. Transient expression of the p35 gene in A.
thaliana protoplasts prevented DNA fragmentation and cell death
upon UV radiation [81]. Transgenic tobacco plants
carrying the gene of protein Op-IAP, a caspase inhibitor, demonstrated
higher resistance to PCD exhibited by suppression of the formation of
dead cell areas during infection with viral or bacterial pathogens [82]. These results suggested that caspase-specific
proteases may exist in plant cells, and that these enzymes may be
involved in PCD and the protective reactions of plants.

Since the natural protein substrate of hypothetical plant caspases was
unknown, the most straightforward way of finding the caspase-like
activity in plants consisted in using peptide fluorogenic substrates
and peptide inhibitors of animal caspases. The canonical peptide
substrates of caspases are tetrapeptides with a XXXD-AFC (AFC,
7-amino-4-trifluoromethyl-coumarin) sequence, where the motif preceding
the amino acid residue D, after which the bond is hydrolyzed by the
enzyme, is typical of each (or several) animal caspase(s). As a result
of enzymatic hydrolysis, AFC is cleaved from the C-terminal aspartate
and starts to emit fluorescence (at 505 nm), allowing the
fluorometric detection of substrate cleavage. The specific peptide
inhibitors of animal caspases have the same XXXD amino acid sequences,
but the aspartate residue is modified by the aldehyde (CHO),
fluoromethyl ketone (FMK), or chloromethyl ketone (CMK) group that
modifies the amino acid residues of the active site (cysteine in case
of caspases) during substrate binding to the enzyme [83].

The peptide substrates of caspases were used for the first time to
reveal the activity of plant apoptotic proteases in 1998 in the classic
publication of del Pozo and Lam [84]. They used
extracts from the NN-genotype tobacco leaves infected with the
tobacco mosaic virus (TMV), where HR was developing, as well as
extracts from uninfected plants. The proteolytic activity hydrolyzing
the specific caspase-1 substrate Ac-YVAD-AFC was detected in the
extracts from apoptotic leaves but not in the extracts from healthy
leaves. Moreover, the specific caspase inhibitors Ac-YVAD-CMK and
Ac-DEVD-CMK could both suppress this YVADase activity and prevent PCD
induced by P. syringae bacterial infection in tobacco plants.
The latter circumstance is particularly significant because it suggests
that the revealed YVADase activity may relate to implementation of the
cell death program.

Dozens of works have been published since that time where various
peptide substrates and animal caspase inhibitors have been tested for
the presence of caspase-like activities in plants. Depending on the
peptide at hand, authors named the revealed activities DEVDase,
VEIDase, YVADase, etc. (see review of Bonneau et al. [6]). Interestingly, the spectrum of detected
caspase-like activities that are revealed during plant PCD caused by
various biotic and abiotic stimuli can be substantially different. For
example, DEVDase activity is registered rather frequently, but in some
cases this activity is absent and another is observed (e.g. YVADase
activity).

Summarizing the results obtained by the described approach (the
application of peptide substrates and animal caspase inhibitors), one
should note that they afforded solid grounds to believe that
caspase-specific proteases are activated in different model systems of
plant PCD, and that the activities of these proteases may be important
for implementation of PCD.

Metacaspases. Soon after the Arabidopsis and rice genomes
were sequenced, it became obvious that plants (at least those with the
sequenced genomes) do not contain caspase genes that could be detected
by simple homology search. Therefore, the above-described caspase-like
activities observed during plant PCD and involved in PCD implementation
seem to be typical of proteases that are structurally different from
caspases. This conclusion was not quite obvious; therefore, the
detection by high-sensitive bioinformatics analysis of two families of
proteases that distantly resembled caspases caused general enthusiasm
[85]. One of these families which is present in
animals and myxomycetes (and, hence, of no immediate interest for us
now) was named paracaspases, and the other one typical for plants,
fungi, and protozoa was named metacaspases.

According to their structure, metacaspases belong to the clan of
cysteine-dependent (CD) proteinases, which also includes caspases (that
cleave peptide bond after a D amino acid residue), legumains (cleavage
after the N residue and, more rarely, N and D residues), separases
(cleavage after an R residue), and bacterial proteases clostripains and
gingipains (cleavage after R and K). These proteolytic enzymes are
combined into a single clan because they have a common type of
three-dimensional structure, the so-called “caspase/hemoglobinase
fold” [86], and they contain a typical dyad
of Cys and His catalytic residues. Plant genomes contain approximately
10 metacaspase genes. Metacaspases are synthesized as precursor
proteins and can be subdivided into two types [87]. Type I plant metacaspases (predominant in some
plants) possess an N-terminal pro-domain that is absent from type II
metacaspases. In the analogy with caspases, metacaspase structure
comprises large (20 kDa) and small (~10 kDa) subunits. In the
case of the precursor protein of type II metacaspases, the small
subunit is separated from the large one by a relatively long linker
sequence. The large metacaspase subunit comprises, as in the case of
caspases, the His and Cys catalytic dyad. Mature metacaspase is formed
through the autocatalytic processing of the zymogen [88, 89]. Metacaspases are
localized in plant cell cytosol (figure).

Diverse localization of proteases involved in programmed cell death in
plants. Metacaspases exist in the cytosol, VPE locates in the vacuoles.
Phytaspases are secreted from the plant cell (by the canonical pathway
including endoplasmic reticulum (ER) and Golgi apparatus) into the
intercellular fluid (apoplast) of healthy plants. On induction of cell
death, the phytaspases are rapidly transferred from the apoplast into
the cytosol

The discovery of metacaspases in plants gave reasonable grounds to
anticipate that metacaspases are the sought caspase analogs in plants
with the specificity and functions of caspases. At first it seemed that
plant metacaspases actually possessed the specificity of animal caspase
hydrolysis. Caspase activity was observed to increase upon
superexpression of the metacaspase genes in plants and to be suppressed
upon RNA interference of metacaspases, as was shown through the use of
fluorogenic peptide substrates of animal caspases [90, 91]. However, everything
fell into place when plant metacaspases were isolated and recombinant
enzymes were obtained, and the autocatalytic processing of metacaspase
precursor proteins was investigated. It turned out that metacaspases
did not hydrolyze the peptide substrates of caspases, but they
possessed strict Arg- and Lys-specificity [88, 92-94]. The subsequent
longstanding discussion in the literature on whether metacaspases could
be considered caspases [95-97] has recently come to an end with the conclusion
that metacaspases are not caspases because they have no aspartate
substrate specificity.

Nevertheless, though metacaspases turned out not to be the
aspartate-specific apoptotic plant proteases that had been sought, what
have we learned about plant PCD during the study of metacaspases? The
modern point of view on plant metacaspases is that metacaspases are
involved in many plant cell processes including PCD. The silencing of
the gene of one of the metacaspases resulted in disturbance of terminal
cell differentiation and embryo development in spruce [91]. Knockouts of the genes of some
Arabidopsis metacaspases displayed no marked disturbance in PCD
implementation, probably due to redundancy (overlapping functions) of
metacaspases. At the same time, metacaspase-8 (AtMC8) superproduction
intensified and RNA interference suppressed the level of PCD caused by
UV radiation and hydrogen peroxide in protoplasts [98]. Seeds and shoots of Arabidopsis plants
with AtMC8 knockout showed enhanced resistance to the herbicide methyl
viologen. The involvement of Arabidopsis metacaspase AtMC4 in
implementation of cell death caused by oxidative stress and Fumonisin
B-1 was described [99]. Recently, it was shown
that type I Arabidopsis metacaspase AtMC1 is a positive
regulator of HR, while another metacaspase (AtMC2) suppresses the
proapoptotic effect of the former [100].
Interestingly, this suppression does not require the presence of
proteolytic activity in AtMC2, because the active site Cys residue
mutant retained the antiapoptotic properties. Thus, metacaspases seem
to be multifunctional cell enzymes.

The only known protein substrate of plant metacaspases is the
evolutionally conservative protein TSN (Tudor staphylococcal nuclease),
the function of which in plants has not yet been established [101]. The recombinant TSN protein is hydrolyzed by
spruce metacaspase mcII-Pa at four sites in accordance with metacaspase
specificity (after the R and K residues). Analogous fragmentation of
this protein is observed during PCD caused by oxidative stress or
occurring during embryo development. In is interesting to note that the
human TSN protein is a target of caspase-3; however, in this case only
one break of regulatory significance is introduced into the TSN
molecule, and it does not coincide with the sites of metacaspase
hydrolysis.

Vacuolar protease VPE. The vacuolar processing enzyme (VPE) of
plants is a cysteine-dependent protease and is localized in plant cell
vacuoles (see figure), where it participates in the processing of
vacuolar proteins. VPE is related to legumains, which belong to the
clan of CD proteases, which includes caspases, metacaspases, and a
number of other proteases (see above). The affiliation of VPE with the
CD clan is demonstrated by three-dimensional fold of the proteolytic
domain and the presence of catalytic residues His and Cys in
characteristic positions. However, the similarity between the amino
acid sequences of VPE and other members of the clan is very low. Like
most proteases, Arabidopsis VPE is synthesized as an inactive
precursor, and the cleavable pro-domains are present at both N- and
C-termini of the protease domain. The precursor protein can be
processed autocatalytically [102, 103]. At the very N-terminus of the precursor
protein, a signal peptide resides that directs the synthesized protein
to the vacuole.

VPE possesses a substrate specificity typical of legumains, and it
hydrolyzes a peptide bond after an asparagine (N) residue; common
inhibitor of this protease is Ac-ESEN-CHO. It has been shown through
the use of a number of synthetic peptides that correspond to some plant
protein sequences that VPE can hydrolyze peptide bonds also after some
D residues, though the efficiency of such cleavage is lower than that
occurring after an N residue [102]. Moreover, one
of the caspase inhibitors (biotinylated VAD-FMK) when introduced into
leaves became covalently bound to VPE [104]. It
is interesting that Ac-YVAD-CHO could act as a competitor of VAD-FMK,
while Ac-DEVD-CHO had no such ability. The binding of
Arabidopsis VPE to VAD-FMK and YVAD-CMK was confirmed by another
research group [105]. Based on these findings,
the authors concluded that plant VPE has caspase-1 activity. The
“partially purified” recombinant γ-VPE which had been
produced in insect cells (one of four VPE forms existing in plants) [106] was able to hydrolyze a fluorogenic substrate
of VPE, Ac-ESEN-AMC (AMC, 7-amino-4-methylcumarin), and caspase-1,
Ac-YVAD-AMC, but it did not hydrolyze Ac-DEVD-AMC (the substrate of
caspase-3).

Another example relates to the Papaver VPE. The recombinant
enzyme was produced in E. coli cells and purified by affinity
chromatography. The enzyme was able to bind biotinylated DEVD-CHO but
not YVAD-CHO. Surprisingly, such activity was common for the precursor
enzyme and the enzyme that preserved the N-terminal pro-domain, but not
mature VPE [107]. Nevertheless, the
Papaver VPE exhibited hydrolytic activity not only towards the
Ac-DEVD-AMC substrate, but also towards the YVAD-derivative, as well as
the derivative of IETD and, to a lesser degree, LEVD and VEID. In these
cases as well, only the precursors but not the mature VPE displayed
proteolytic activity. Another unexpected result was that the proVPE
that displayed the activity did not undergo autocatalytic
processing.

Thus, at present it is not quite clear which caspase-like activity VPE
possesses and whether the processing of the precursor protein is
required for the activation of the enzyme and how it may occur. The
existing discrepancies are probably associated with differences between
species or with the methods of isolation of the enzyme for the analysis
of its activity. Nevertheless, is VPE related to plant PCD? The
available data demonstrate that VPE may be involved in implementation
of PCD, and it is associated not least with the vacuolar localization
of this enzyme (see figure). VPE has been shown to take part in PCD
that occurs in tobacco leaves during viral (TMV) infection and includes
the breakage of the tonoplast, DNA fragmentation, and formation of dead
cell areas. The silencing of four VPE genes suppresses the collapse of
the vacuole, DNA fragmentation, and formation of necroses [74, 104, 108]. How exactly VPE may be involved in the
described processes remains unknown. Interestingly, expression of the
VPE genes increases in the beginning of HR and then declines rapidly.
This effect is probably indicative of the role of VPE at the early
stages of cell death. VPE is also involved in PCD induced by some
fungal toxins. Morphologically, this cell death is similar to
TMV-induced HR [106]. In this case, inactivation
of all four VPE genes also suppresses PCD.

It should be noted that inhibition of VPE (by Ac-YVAD-FMK, in
particular) had no effect on development of PCD in response to
bacterial infection, in contrast to PCD caused by viral infection [75]. At the same time, it is worth mentioning that
the fusion of the vacuolar and plasma membranes that takes place during
PCD caused by certain pathogenic strains does not depend on VPE
either.

Thus, a relationship between VPE activity and some forms of plant PCD is
evident. Elucidation of the extent to which this involvement of VPE in
cell death may be associated with the assumed caspase-like activity of
the enzyme and finding of the apoptotic protein targets of VPE is in
prospect.

Subtilisin-like plant proteases with aspartate specificity. The
alternative approach to the search of PCD-related plant proteases based
on identification of a plant protease that hydrolyzes the native target
protein at the same site as the animal caspase proved to be efficient.
The VirD2 protein of the agrobacterium Agrobacterium
tumefaciens – a plant pathogen, was shown to be
specifically fragmented by human caspase-3. It then was shown that
induction of HR in Nicotiana tabacum plants of the NN
genotype caused by TMV infection is accompanied by activation of a
plant protease with a similar specificity in hydrolysis of the VirD2
protein (protein cleavage after the D residue in the TATD motif) [109]. The revealed protease was named phytaspase
(from “plant aspartate-specific protease”)
[110]. Phytaspase activity may also be registered
during mechanical destruction of plant tissue, which has revealed
phytaspase activity in quite different plants [111] including dicotyledons and monocotyledons.

Identification of tobacco and rice phytaspases demonstrated that
phytaspases are subtilisin-like proteases (subtilases) of plants [110]. Although it had been assumed that caspase-like
(in the functional sense) plant proteases could be structurally
different from animal caspases (otherwise, phytaspases would be
identified long ago by homology), it was hard to expect that the
difference would be so drastic. Indeed, the structure of
subtilisin-like proteases is dramatically different in structure from
caspases. Subtilases are Ser-dependent proteases, while caspases are
Cys-dependent. An active caspase is a tetramer consisting of two large
and two small subunits, while phytaspase is a monomer. The presence of
a potential signal peptide in the precursor protein of phytaspases (see
below) could be indicative of extracellular localization of the enzyme,
while caspases are intracellular proteins (see table).

Comparison of properties of aspartate-specific apoptotic animal and
plant proteases: caspases and phytaspases

Nevertheless, the study of the substrate specificity of tobacco and rice
phytaspases has shown that these enzymes, just as caspases, hydrolyze
substrates strictly after a D residue in a certain amino acid context
[110]. The optimal substrate of phytaspases is
the Ac-VEID-AFC peptide (substrate of caspase-6), though a comparable
(2-4 times lower) rate of hydrolysis was observed for the derivatives
of VAD (substrate of various caspases), YVAD (substrate of caspase-1),
VDVAD (the substrate of caspase-2), IETD (the substrate of caspase-8),
and LEHD (substrate of caspase-9) (table). The only exception was
Ac-DEVD-AFC (substrate of caspase-9), which was not hydrolyzed by
phytaspases at all.

The ability of phytaspases from different plant organisms to hydrolyze
various peptide substrates may create an illusion of a relatively low
selectivity of the plant enzyme. However, such conclusion seems to be
erroneous on taking into consideration the fact that phytaspases exceed
the human caspase-3 in selectivity at the level of protein substrates.
Phytaspase is undoubtedly a “processing”, not a
“digestive” proteolytic enzyme. However, this statement
concerns also animal caspases.

Phytaspase with its broad spectrum of hydrolyzed peptide substrates of
caspases alone is able to explain almost the entire diversity of
caspase-like activities revealed during plant PCD on such substrates.
Therefore, the number of various caspase-like enzymes in plants is
probably not oppressively large, as it was commonly supposed. Yet,
there is an exception. Phytaspase has no DEVDase activity (table),
which is revealed quite often during PCD in plants. Therefore, it may
be anticipated that at least one caspase-like protease of plants has
not yet been discovered.

Phytaspases were shown to participate in plant PCD caused by biotic and
abiotic stresses. Cell death was stimulated by the enhanced level of
phytaspase activity during superproduction of the enzyme, while
reduction of phytaspase activity by a specific inhibitor or through RNA
interference suppressed PCD [110].

Thus, phytaspases are similar to caspases both in their specificity and
in the role of these proteases in PCD. However, phytaspases are
fundamentally different from caspases in structure, and this difference
has important functional consequences. It has been shown that
phytaspases are synthesized as inactive precursor proteins that contain
an N-terminal signal peptide, pro-domain, and the protease domain. The
N-terminal signal peptide as a part of the proenzyme directs phytaspase
secretion from a plant cell. Active phytaspase is formed through
pro-domain cleavage. This process is autocatalytic and constitutive,
i.e. it occurs even in the absence of PCD. Pro-domain cleavage is
required for the formation and secretion of the proteolytically active
enzyme.

In healthy plant tissues, phytaspase accumulates in the intercellular
fluid (apoplast) (see figure). Thereby, spatial uncoupling of the
enzyme and its intracellular substrates is achieved. However, upon the
induction of PCD, the phytaspase is re-localized from the apoplast into
the dying plant cell and gains access to its intracellular target
proteins [110]. The mechanism of this absolutely
novel phenomenon is unknown, but there are grounds to believe that
“retrograde” transport of phytaspase occurs
specifically.

Localization of the processed phytaspase in the apoplast may imply that
the enzyme has protective functions associated with proteolysis
(neutralization) of the effector proteins of bacterial pathogens. The
only natural target protein of apoptotic plant subtilases known at
present is the protein VirD2 of the phytopathogenic bacterium A.
tumefaciens. When infecting plants, the bacterial protein VirD2
with attached bacterial DNA (T-DNA) enters the cytoplasm and then is
imported into the nucleus of the plant cell. This provides the
integration of bacterial DNA into the plant genome and plant cell
transformation [112]. For active transport into
the nucleus, the VirD2 protein, despite being bacterial, is equipped
with a nuclear localization signal [113]. It has
been shown that the hydrolysis of agrobacterial VirD2 protein by
phytaspase is a protective mechanism of plant cells limiting the
delivery into the nucleus and expression of foreign (agrobacterial) DNA
[114]. This is due to the fact that VirD2
cleavage by phytaspase at the TATD400 site results in removal of a
short C-terminal fragment of the VirD2 protein. Since the nuclear
localization signal of VirD2 is located in this very region, VirD2
loses the ability to be imported into the plant cell nucleus and to
carry there the attached bacterial T-DNA.

Subtilisin-like proteases of the S8A subfamily which includes
phytaspases and all other plant subtilases are usually not
characterized by high specificity of hydrolysis [115, 116]. The ability to
specifically hydrolyze substrates strictly after the D residue is
generally uncommon for proteases. In this respect, it is worth noting
that each plant species has dozens of subtilase genes (more than 50
family members for A. thaliana and more than 60 for rice
Oryza sativa [117, 118]). Just a few representatives of these families
have been characterized, and none of the previously identified
subtilases had a phytaspase activity. It is an interesting question how
many members of the subtilase family in each plant species have
aspartate specificity: one or several? If several such proteases exist,
it would be interesting to know whether these enzymes differ in the
preferred sites of hydrolysis, localization, expression in tissues, and
involvement in the process of PCD during development and in response to
stresses.

Phytaspases display similarity with the animal and yeast subtilisin-like
proteases, so-called proprotein convertases, which belong to the S8B
subfamily (which apparently is absent from plants), in the high
selectivity of substrate hydrolysis [119, 120]. Convertases are involved in the processing of
precursor proteins, which results in formation of biologically active
peptides and proteins, and one may assume an existence of a similar
function of phytaspase in the “out-of-PCD” hours. It should
be noted, however, that convertases introduce a break into target
proteins after the basic amino acid residues (K, R) but not after D.

Plant subtilisin-like proteases apparently include also the saspase of
oat [121]. The PCD model that helped to discover
this activity was the oat Avena sativa plant infected by the
fungus Cochliobolus victoria. The pathogen secretes an unusual
toxin named victorin. Victorin-induced PCD is a HR to the infection [68, 122, 123]. The protease isolated from the oat leaves
treated with victorin was named saspase (serine-dependent
aspartate-specific protease). Saspase, just as phytaspase, is able to
hydrolyze many peptide substrates of caspases [121, 124] and, at the same
time, lacks the DEVDase activity. Several peptides of the enzyme were
sequenced showing that the protein must be a plant subtilisin-like
protease (subtilase). One may think that saspase is an oat phytaspase,
but there is an interesting difference between the two classes of
enzymes. Whereas phytaspases of two evolutionally distant plants
(tobacco and rice) prefer the VEID derivative as a substrate, saspase
does not hydrolyze VEID derivative at all [121].
The reason for this difference can be elucidated after identification
of saspase.

Interestingly, saspase activity became detectable in the apoplast under
PCD induction by victorin or heat shock, though before PCD induction
such activity had not been observed in the apoplast [121]. Presumably upon PCD induction either rapid
secretion of saspase into the apoplast or unmasking of the protease
already present in the apoplast may occur.

CONCLUSION

PCD in plants displays quite a number of common features with PCD in
animals but, on the other hand, it has a number of substantial
differences. Proteolytic enzymes that are involved in PCD are localized
in different compartments of plant cells: the cytoplasm (metacaspases),
the vacuoles (VPE), and the intercellular fluid (phytaspases) (figure).
However, the example of phytaspase which is being transferred from the
apoplast into the cytoplasm upon PCD induction shows that localization
of the enzyme in a certain compartment of healthy cells (tissues) does
not exclude the functioning of this protease in a quite different
compartment of dying cells. It is of considerable interest how the
functions are distributed among the plant apoptotic proteases and
whether they can influence each other’s function.

Among the proteolytic enzymes of plants described above which are
involved in PCD, phytaspases most closely correspond to animal caspases
by substrate specificity (table). At the same time, the apoptotic
proteases of animals and plants are totally different in structure and,
moreover, phytaspases are Ser-dependent, while caspases are
Cys-dependent enzymes. The comparison of caspase and phytaspase
properties gives the impression that animals and plants may have
followed different tactics that eventually resulted in a similar policy
decision: creation of proteases with similar function and
specificity.

Animals and plants use different strategies with respect to their
apoptotic proteases. Both caspases and phytaspases are synthesized as
inactive precursor proteins; however, further their paths diverge.
Procaspases are stored within animal cells. They are activated through
the processing and association of subunits in response to PCD-inducing
stimuli, which results in fragmentation of intracellular target
proteins and cell death. In contrast to this scenario, prophytaspases
are processed constitutively and autocatalytically, forming active
enzyme even in the absence of a PCD-inducing stimuli. However, mature
phytaspases are secreted from the cell into the apoplast (due to the
presence of signal peptide in the precursor protein). It allows the
active proteolytic enzyme to be spatially separated from intracellular
target proteins so that unauthorized proteolysis and cell death can be
avoided. Under PCD induction, phytaspase is transported from the
apoplast into the cells, which results in fragmentation of
intracellular proteins.

Thus, intracellular caspase activity is controlled at the level of
processing of precursor proteins, while phytaspase activity is
controlled at the level of enzyme transport from the apoplast into the
cytoplasm. It may be concluded that plants have developed their own
mechanism to control apoptotic proteases absent from (or not yet
discovered) in animals [124, 125]. Thus, animal and plant cells demonstrate both
common features and substantial differences in how they treat their
apoptotic proteases.

One of the basic approaches for elucidation of the mechanism of action
and new functions of PCD-related plant proteases is the identification
of the target proteins of these enzymes. The study of cellular protein
substrates will disclose important features in the molecular mechanisms
of plant PCD, reveal novel signaling pathways, and allow more thorough
comparison of the animal and plant machineries responsible for the key
processes of cell life and death.

Work in the authors laboratory was supported by the Russian Foundation
for Basic Research (projects No. 11-04-01120 and No. 11-04-00984) and
by the Ministry of Education and Science of the Russian Federation
(P334, 14.740.11.0168).